Drug and pharmaceutical composition for the treatment of lesions of the digestive tract

ABSTRACT

Use of at least one polymer or one biopolymer, called HBGFPPs, specifically protecting the growth factors of families of FGFs and beta TGFs from tryptic degradation and not significantly inhibiting coagulation, in the manufacture of a drug for the treatment of lesions of the digestive tract and of primary or secondary derived tissues of the endoderm and the mesoderm.

FIELD OF THE INVENTION

This invention relates to the use of polymers or biopolymers for thepreparation of a drug for the treatment of lesions of all originsaffecting the digestive tract from the buccal cavity to the analextremity in human or veterinary medicine.

It also relates to a composition containing these polymers and intendedfor such a treatment.

BACKGROUND

The synthesis of CMDBS polymers (dextrans substituted by carboxymethyl,benzylamine and sulfonate) has been described in French Patent 2 461 724and U.S. Pat. No. 4,740,594. Some of these polymers mimic heparin andmay be used as plasma heparin replacement products, thanks to theiranticoagulant and anticomplement properties.

Some of these CMDBS polymers mimic another property of heparinconsisting of a stabilization, protection and potentialization of the invitro biological activity of the growth factors of the FGF family(Tardieu and coll., Journal of Cellular Physiology, 1992, 150 pp. 194 to203).

French Patent 2 644.066 describes the use of certain CMDBSs associatedwith FGFs for healing the skin and the cornea. Experiments have beenconducted by provoking a cutaneous wound with the help of a hollow punch6 mm in diameter in the rat. In this example, the CMDBS associated withthe FGF 2 makes it possible to obtain a definite effect on the speed andthe quality of skin repair.

Another biopolymer, dextran sulfate, has also been advanced inassociation with FGFS, as a stabilizer and protector, in Japanese PatentNo. 13890. Dextran sulfate, moreover, is widely used in skin healingointments and creams as well as in collyrium compositions, but, to theknowledge of the applicant, has no reported effect on the healing andregeneration of muscular lesions.

Another agent, sucrose sulfate ester and its aluminum salt, sucralfate,are products described and used, on their own or associated with FGFs,as agents in the treatment of ulcers and lesions of the digestive tract(U.S. Pat. No. 3.432.489 ) and in different pharmaceutical associationsand compositions described in a series of patents (U.S. Pat. Nos.4,975,281, 4,885,281, 5,013,557, 5,164,379, 5,196,405, 5,240,710 andDanish Patents 102488 and 505588).

The tissues of the digestive tract are particularly rich in growthfactors and several authors have described the presence and/or action ofFGFs and beta TGFs in/on enterocyte cells or the healing action of thesefactors in lesions of tissues of the digestive tract as well as thepresence or the action of growth factors presenting an affinity forheparin or heparan such as AB or BB PDGFs or Hepatocyte Growth Factor(Lemoine N R; Leung H Y; Gullick W J; Growth Factors in theGastrointestinal tract ; Gut 1992, 33, pp. 1297 to 1300; Di Gulietta A,Hervada T; Nardy R V; LESH C A: Effect of platelet derived growth factorBB on indomethacin-induced gastric lesions in rats; Scand. J.Gastroenterol. 1992, 27, pp. 673 to 676; Tajagascu M and coll.Hepatocyte growth factor induces mitogenic reaction to the rabbitgastric epithelial cells in primary culture; Biochem. and Biophys. Res.Comm. 1993, 191; 528-534; Mustoe and Coll. Differential acceleration ofhealing of surgical incisions in the rabbit gastrointestinal tract byplatelet derived growth factor and transforming growth factor beta;Surgery 1990, 108, pp. 324 to 330; Katayama M; Kan M: Heparin-binding(fibroblast) growth factors are potential autocrine regulators ofoesophageal epithelial cell proliferation, In Vitro Cell. Dev. Biol.1991, 27, pp.533 to 541).

It thus emerges from an analysis of the state of the prior art thatpolymers have already been used in association with growth factors oncertain lesions of a very particular type of tissue, namely cutaneoustissue.

In view of the unpredictable nature of the therapeutic effects of agiven molecule, it was not clear wether these polymers could have aneffect on tissues other than those of the skin.

It is, in fact, well known that the different tissues of the human oranimal body present structural and functional specific features makingit impossible to predict the effect of a molecule, known for its effecton the cutaneous tissue, and on tissues of the digestive tract.

This is particularly true in view of the fact that the tissues of thedigestive tract and those of cutaneous tissues, for example, havedifferent embryonic origins.

Similarly, it is well known that it is impossible to predict the in vivoactivity of a molecule on a particular tissue from results obtained invitro on a specific experimental model.

All the other known drugs in this field, such as anti ulcerous drugs,act by protecting the mucosa by antacid gels, by inhibition of gastricacid secretions or by H₂ anti-receptor action.

The activity of a mixture of glycosaminoglycans, mesoglycan, onhemorrhoids has been tested by Saggioro et al., (1985, Min. Diet. e.Gastr, 31, pp. 311-315). This publication shows that mesoglycan is veryeffective against hemorrhoids, eliminating the symptoms while improvingthe endoscopic aspect.

However this publication teaches only its inquiry polymers of a veryparticular type which, moreover, present a composition of poorly definedanimal origin and subject to variations.

SUMMARY OF THE INVENTION

Surprisingly, it has been found, according to the invention, thatcertain polymers have a very marked effect on the speed of healing andregeneration of lesions of digestive tract tissues as well as on thequality and solidity of these scars.

It has also been shown that very low doses of these polymers providetherapeutic effects.

This invention relates to the use of at least one polymer or onebiopolymer, called HBGFPP, with the exception of mesoglycan,specifically protecting the growth factors of the FGF and beta TGFfamilies from tryptic degradation and not significantly inhibitingcoagulation, in the manufacture of a drug for the treatment of lesionsof the digestive tract, and of secondary and primary derived tissues ofthe endoderm and the mesoderm.

In particular, such a polymer displays an anticoagulant activity of lessthan 50 international units per mg of polymer, measured according toMaillet et al. (Mol. Immunol, 1988, 25, 915-923). Advantageously, thepolymer potentializes the FGFs in vitro.

Preferably, it does not substantially activate the complement system,that is to say, it possesses an anti-complement system of above 0.5 μgfor the CH₅₀ (according to Mauzac et al., Biomaterials, 6, 61-63, 1985).

BRIEF DESCRIPTION OF THE DRAWINGS

According to the invention, polymers are understood to mean any naturalsubstance, chemically modified natural substance or totally syntheticsubstance responding to the definition given above.

The following polymers are therefore concerned:

polymers obtained from dextrans but modified by other types ofsubstitutions with other types of radicals,

natural polymers other than those deriving from dextrans but includingosidic residues (cellulose, chitin, fucanes, etc.),

polymers obtained by polymerization of monomers of non-osidic nature(malic polyacid, oxalic polyacid, lactic polyacid, polystyrene,polyethylene glycol), whether or not modified.

Advantageously, the said polymer or biopolymer is a polysaccharide whichmay be principally composed of glucose residues.

It may also comprise glucosamine and/or uronic acid residues,particularly in the form of glucosamine dimer-uronic acid.

Such a polysaccharide advantageously displays a molecular weight of atleast 10 kD and advantageously of about 40 kD.

This invention also relates to a pharmaceutical composition containingthese polymers.

The polymers and/or biopolymers may be selected from natural substanceswhich may then be modified, if required, by additions of appropriatechemical groups, or again be obtained entirely by synthesis. Thesenatural, semi- or wholly synthetic polymers are then selected on thebasis of their ability to interact specifically with several growthfactors, notably those of the FGF and the beta TGF families. They arealso selected on their ability to protect this (or these) factor(s)against proteolytic degradations. These polymers will be referred tounder the generic abbreviation HBGFPP (heparin binding growth factorprotectors and promoters). Two prototypes of these polymers orbiopolymers are given as examples together with the processes andselection criteria of these polymers.

The first HBGFPP example belongs to the CMDBS family which are knownproducts, namely functionalized biospecific dextrans, substituted bycarboxymethyl, benzylamide and benzylamine sulfonate. These polymersillustrate the yielding of HBGFPPs from natural products (dextrans)which are subsequently chemically substituted. The second exampledescribes the selection of wholly natural products, such as purifiedsulfate proteoglycosaminoglycans from tissular extracts.

These two examples illustrate the ability of these HBGFPPs to interact,stabilize, protect and potentialize the growth factors of the FGF andbeta TGF families, and their use in a pharmaceutical compositionpermitting the protection, healing and/or regeneration of lesions of allorigins affecting the digestive tract from the buccal cavity to the analextremity.

In this patent application, by <<treatment>> is meant any curative orpreventive operation carried out for the prophylaxis and the healing oflesions of the digestive tract, whether these lesions be of superficialor profound ulcerous type and whatever their origin, and/or the healingof perforations and/or surgical cuts and incisions as well asanastomoses performed on appropriate regions of the digestive tract.

A drug or a pharmaceutical composition according to the inventioncontains an effective amount of HBGFPP, for example CMDBS associatedwith one or more compatible and pharmaceutically acceptable vehicles. Itmay also be associated with agents such as, for example,anti-inflammatory, antibacterial, antifungal agents, gels, plasters,antacid dressings, H₂ anti-receptor agents or anti-proton pump agentsused in the treatment of gastric ulcers, and agents commonly used inbuccal or dental care or in the treatment of hemorrhoids.

Advantageously, such a composition is designed to be absorbed directlyby oral route, deposited on the lesion or injected if the said lesion isdirectly accessible, notably in subcutaneous, buccal or rectal lesions,or again during surgical interventions by depositing or soaking theextremities of the tissues before restitching or repatching or carryingout anastomoses. The unit dose is from 10 to 2500 μg of CMDBS or HBGFPPper ml.

The vehicle may be physiological serum or buffers, such as PBS,containing 0.15M NaCl or any other compatible solution which does notirritate the damaged tissue. Formulations providing thick solutions orin gel or aerosol according to standard techniques known to the personof ordinary skill in the art may be proposed depending on the type andthe accessibility of the lesion.

Thus, for directly accessible lesions, such as buccal aphthas, lesionsof the tongue or the palatal vault, or of gums and all support tissuesof teeth or of the throat, the composition may be applied in the form ofa solution, suspension, spray, powder, gel, unguent or ointment, pasteor gelatinous cream, toothpaste, dental fixative, periodontal implant,chewing gum, effervescent tablets or tablets to be sucked, and may alsobe applied by means of a little pipette, spatula or brush. Thecomposition described in this invention has the effect of curing thelesions, whether these lesions are accidentally caused, as in the caseof thermal or chemical cuts and burns of microbial, fungal or viralorigin (provided that the cause of the lesion is also treated byassociating antimicrobial, antifungal or antiviral agents), or whetherthe lesions are due to other origins which are not always identifiableor known, or lesions resulting from chemotherapy or radiotherapy.

Similarly, for anorectal lesions, diseases or irritations, thecomposition concerned by this invention provides a treatment whichreduces the irritation, itching and swelling of the tissues, and thesuffering caused by anorectal diseases, and leads to the curing of theselesions by favoring tissue repair and regeneration. The anorectaldiseases involve the perianal regions, the anal canal and the rectum andinclude hemorrhoids of extremely varied origin such as constipation,diarrhea, pregnancy, anal infections, rectal carcinomas, and otherdisorders like anal fistulas and fissures which may have diverse causessometimes induced by chemotherapy or radiotherapy. As with lesions ofthe upper passages of the digestive tract, anorectal lesions aredirectly accessible and the composition may be applied in the form of asolution, suspension, spray, powder, gel, unguent or ointment, paste orgelatinous cream, or a suppository and by using a pipette or syringewhose nozzle is introduced in the anal canal or a spatula, brush or anyother appropriate means.

The composition may be associated with other compositions commonly usedin the treatment of anorectal diseases which have been listed nonexhaustively in a monograph, No. 45 35576 the May 26, 1980, published bythe American Food and Drug Administration (FDA). This monographdescribes over 75 ingredients, classified in families such as localanesthetics, vasoconstrictors, protectors, anti-irritants, astringentagents, healing agents, antiseptics, keratolytics and anticholinergics.

The results obtained by applying this composition are of a quitedifferent order to those obtained with existing products includingsucralfate-based compositions such as those described in U.S. Pat. No.5,196,405 issued on the of Mar. 23, 1993 for the treatment ofhemorrhoids or in U.S. patent application Ser. No. 52407010 (DK102488and DK505588). Depending on the surface of the wound, the CMDBS or HBGFPproduct doses correspond to a fraction of an initial solution of 10 to2500 ug per milliliter (which, for a local application on common wounds,implies a dose which is rarely above a few hundred microliters ofsolution), this dose being applied once or twice per 24 hours. Thesucralfate compositions used are 0.01 to 5% (according to U.S. Pat. No.5,196,405), in other words at least 10 times higher than those describedin this invention, and the effects described in all the examples of thesaid U.S. Pat. No. 5,196,405 are obtained from 50 milligram permilliliter compositions.

For digestive tract lesions which are not easily accessible by topicalroute, the administration route of the composition is either oral orrectal. This HBGFP-based composition is effective in the treatment oflesions of the pharynx, the stomach, the esophagus, the duodenum, thesmall intestine and the colon. Mention may be made, as non exhaustiveexamples of lesions which may be treated with benefit by the HBGFP-basedcompositions concerned by this invention, of mucosa lesions of parts ofthe digestive tract provoking ulcers such as gastric acid or duodenalulcers; lesions induced by drugs, by accidentally absorbed chemicalproducts, or by treatments by radiation, lesions induced by stress or byfoodstuffs, or of surgical or traumatic origin, viral lesions or thoseassociated with inflammations such as hemorrhagic rectolitis, or Crohn'sdisease.

The stimulating activity of the reparation of digestive tract tissuesmay also be envisaged for other secondary and primary derived tissues ofthe endoderm and the mesoderm such as glandular derivatives, for examplethe liver, the pancreas, the salivary glands, the adenohypophysis, orpharyngeal and respiratory derivatives, for example bronchi, lungs,pleuras.

The HBGPPs may thus be recommended in the reparation of lesions of allorigins affecting these other secondary and primary derived tissues ofthe endoderm and the mesoderm (traumatic lesion induced by surgery or bytumoral or other pathologies).

FIG. 1 represents the CMDBS formula.

FIGS. 2A and 2B illustrate the potentialization of the biologicalactivity of FGF1 (2A) and FGF2 (2B) by heparin, mesoglycan andsulodexide. Biological activity is measured on CCL39 cells by measuringthe increased incorporation of tritiated thymidine versus the dose ofFGF1 and FGF2 added alone or in the presence of 20 μg of heparin, 10 μgof mesoglycan or 10 μg of sulodexide.

FIGS. 3A, 3B, 4A and 4B illustrate the protective effect of heparin,mesoglycan and sulodexide against a thermal degradation of FGF1 (3) andFGF2 (4) . FGF samples are incubated on their own or in the presence of20 pg of heparin, 10 μg of mesoglycan or 10 μg of sulodexide at 20° C.(a) and 37° C. (b) for 1, 7, 15, 30 days. The measurement of thebiological activity presented in abscissa corresponds to the stimulationunit values (ED₅₀) of the incorporation of tritiated thymidine in CCL39cells.

FIG. 5A illustrates the protective effect of heparin, mesoglycan andsolodexide against a proteolytic degradation of the ¹²⁵ I-FGF1.Proteolytic digestion was carried out at 37° C. and the samples wereseparated by 18% polyacrylamide gel electrophoresis. The gels are driedand autoradiographed. The first track contains the 125I-FGF1 on its own.The ¹²⁵ -FGF1 is incubated in the presence of trypsin (track 2), heparin(track 3), mesoglycan (track 4) or sulodexide (track 5).

FIG. 5B illustrates the protective effect of heparin, mesoglycan andsulodexide against a proteolytic degradation of the ¹²⁵ I-FGF2. Thearrangement of the tracks is identical to that presented for the ¹²⁵I-FGF1 in FIG. 5A.

FIGS. 6A and 6B are DEAE-Trisacryl column elution profiles,respectively, of HSM fractions (FIG. 6A) and HSS fractions (FIG. 6B) inthe presence of chondroitin sulfate (CSA) fractions for the calibrationof the column.

EXAMPLE 1

CMDBS preparation and selection

a) CMDBS preparation

CMDBSs are dextrans substituted by carboxymethyl, benzylamide andbenzylamide sulfonate groups. The method of synthesizing the CMDBSs maybe that described by M. Mauzac and J. Josefonvicz in Biomaterials 1984,5, pp 301-304.

According to this process, carboxymethyl dextran (CMD) is prepared fromdextran by substituting several glycosylated units with carboxyl groupson the carbon in positions 5 and 6.

In the next phase, benzylamide is coupled with the carboxyl groups toform carboxymethyl-benzylamide dextran (or CMBD). Finally, a fewaromatic nodes of benzylamide are sulfonated in order to yieldcarboxymethyl dextran benzylamide sulfonate or CMDBS.

The sodium salts of these derivatives are ultrafiltered, lyophilized anddissolved in the appropriate buffer prior to use.

The general formula of the CMDBSs is illustrated in FIG. 1.

The CMDBSs possess a statistical distribution of the differentsubstituents. The percentages for each CMDBS type are determined byusing standard methods.

b) CMDBS selection

i: FGFs protection and stabilization tests

During the synthesis of the CMDBS the substitution rate of each of thegroups may be controlled by modifying the substitution reactionconditions. Control of such parameters as temperature, reaction time,relative concentrations of the constituents, substitution reactionnumber, etc., makes it possible to obtain a very large number ofsubstituted polymers. The substitution of the hydroxyls by carboxymethylon the carbons in positions 5 and 6 gives carboxymethylation ratesranging from 0 to 200% (100% for each of the carbons in position 5 and6). The carboxymethyl group may, in turn, be partially or totally usedfor fixing the benzylamide. The benzylamide groups may be partially ortotally used for the sulfonation. The functionalized substituteddextrans used according to the invention are among those specificallydescribed in French Patent 2.461.724. In addition to its ability tostabilize and protect FGF family growth factors, as described in thepublication of Tardieu et coll., J. Cell. Physio. 1992, 150, pp 194 to203 and in French Patent 2.461.724, the selected CMDBS must be able tointeract with at least one member of the growth factors family belongingto the beta TGF family according to an evaluation method describedbelow, and to protect the beta TGFs against proteolysis.

ii. Evaluation of the interaction capacities between CMDBS and beta TGFfamily growth factors.

In order to measure the capacity of certain CMDBSs to interact withmembers of the beta TGF family and, by means of this interaction, toprotect the beta TGFS, a grading test was devised. This test consists inmeasuring the ability of the selected CMDBS to allow the beta TGF tomaintain its biological activity despite a protease treatment.

In the example below, the CMDBS used is batch 26.2 defined by asubstitution rate of 110% of carboxymethyl units, 3.6% of benzylamideunits and 36.5% of sulfonate units, and possesses an anticoagulantactivity of 4 IU/mg (International Units). This batch's anticomplementactivity is 1.1 μg of CH₅₀ measured according to Mauzac et al.(previously cited).

The heparin used as control was supplied by the Sanofi company (ChoayInstitute) and presents an anticoagulant activity of 175 IU/mg.

The beta 1 TFG is prepared from human blood platelets according to aprotocol described in numerous publications (for example Growth factorsand their receptors, 1992, vol 1 pp 419-472, written by A. Roberts andM. Sporn, edited by A. Roberts and M. Sporn, and published by SpringerVerlag Berlin) and is commonly used by persons of ordinary skill in theart. The beta TGF biological activity test used in this example is thatof the inhibition of CCL64 cells (from the American Tissue CultureCollection). This inhibition is measured by the ability of the beta TGFto inhibit the incorporation of tritiated thymidine in a dose dependentmanner in these CCL64 cells stimulated by the FGF or by fetal calf serumaccording to the protocol described by Van Zolen in Progress in GrowthFactor Research, 1990, 2, pp 131 to 152. Two doses of beta TGF are used,one corresponding to the 50% inhibition capacity of the incorporation oftritiated thymidine (defined as the inhibiting activity unit) and theother corresponding to the 100% inhibition capacity. In this example,the values obtained are 250 pg of TGF for the CCL64 cells cultivated in1 ml of culture medium.

A 50 ng sample of beta TGF in saline phosphate buffer containing 0.1%bovine albumin serum (from the SIGMA company, Saint Louis USA) isincubated on its own, or associated either with 5000 μg of CMDBS or 5000μg of heparin, with or without 500 μg of trypsin. The final volume ofthe incubated solution is adjusted to 1 ml and incubation is carried outat 37° C. for a varying length of time (10 minutes in the exampledescribed (Table 1)).

20 μl samples of a volume of are taken from each of the incubationreactions and added to CCL64 cells cultivated in 24-well plates, eachwell containing one milliliter of culture medium according to theaforementioned protocol described by E. Zohlen. In these conditions, thefinal concentration of beta TGF per well is 1 ng/ml. Table 1 summarizesthe results obtained in various conditions and shows the protectiveeffect of the CMDBS. Thus, after 10 min of incubation at 37° C., 75% ofthe biological activity of the beta TGF is still present, whereasheparin, despite the fact that it can be fixed to the beta TGF (MacCaffrey et al., J of Cell. Physiology, 1992, vol 52, pp 430-440), doesnot protect the beta TGF against this proteolytic degradation (less than20% of biological activity remain). It should be remembered that, in thecase of FGFs, heparin provides protection against proteolysis induced bytrypsin (Tardieu et al., Journal of Cellular Physiology, 1992, 150: pp.194-203).

It was verified that the CMDBS had no inhibiting power on the activityof trypsin (Table 2). Thus, 10 μg of trypsin were incubated, either witha substrate (S.87 supplied by the Serbio company in Paris and usedaccording to the supplier's recommendations) or with this substrate anda trypsin inhibitor such as that originating from soya (such as thesoybean trypsin inhibitor or STI from Sigma), these incubations beingcarried out in the absence or presence of varying quantities of CMDBS(batch AM26). The enzymatic activity of trypsin was measured byspectrophotometric absorption of the transformation product of S 87versus incubation time.

EXAMPLE 2

Selection of other HBGFPPs

A commercial preparation of proteoglycosaminoglycan andglycosaminoglycans, sulodexide, was selected according to its ability tointeract with the growth factors of the FGF and beta TGF families.

Preparations of heparan sulfate obtained by fractionating mesoglycan andsulodexide were also tested.

The mesoglycan and sulodexide were supplied by the Sigma Chemical Co,Saint Louis Mo. USA. Their properties are summarized in Table 3.

The cells used in this example are CCL39 cells from the American TissueCulture Collection. The conditions concerning culture and themeasurement test of FGF biological activity are the same as thosedescribed in the publication by Tardieu and coll. in the Journal ofCellular Physiology, 1992. The FGF growth factors used are the FGF 1 andFGF 2 recombinant forms.

a) Effect of sulodexide on the in vitro biological activity of the FGFs

In these experiments, the FGF 1 or 2 is used at a dose corresponding tothe effective dose (indicated ED₅₀) in order to induce a stimulation ofthe biological activity of 50% of the dose inducing the maximumstimulation. The biological activity is measured by the ability toinduce an increase of the incorporation of tritiated thymidine in thecells according to protocols extensively described in numerouspublications such as the previously mentioned publication by Tardieu etcoll. and also in French Patent 2 644 066.

In this example, the ED₅₀ is 5 ng/ml for the FGF 1 and 3 ng/ml for theFGF 2, these values being measured experimentally (FIG. 2A and 2B).Thesame stimulation experiment in function of the FGF dose is carried outin the presence of 10 μg/ml of sulodexide, or 20 μg/ml of heparin. FIG.2 shows that in these conditions the ED₅₀ becomes 0.4 ng/ml and 0.2ng/ml respectively for FGF 1 and FGF 2 in the presence of these doses ofsulodexide or heparin. In addition to this ability to potentialize thebiological activity of the FGFs, the HBGFPPs protect the FGFs againstthermal degradations and the inactivation induced by the proteolyticaction of trypsin (FIGS. 4 and 5). Similarly, these HBGFPPs protect FGF1and 2 against an inactivation induced by the proteolytic activity oftrypsin (FIGS. 5A and 5B).

b) Protective effects of sulodexide, dextran, dextran sulfate andsucrase with regard to beta TGFs.

Several other compounds were evaluated: dextran sulfate (Sigma Chemical,molecular weight 40.000, the dextran having been used for the synthesisof the CMDBS (also from Sigma)), sucrase or sucrose octasulfate(supplied by D. Bar Shalom, Bukh Medic company, Denmark). Some of thesecompounds were chosen because they protect and stabilize FGFs, forexample sucrase (see. U.S. Pat. No. 5,202,311) or dextran sulfate (see.Japanese Patent 138 907/88). The dextran is the one used in thesynthesis of CMDBS AM26.

The protection experiment of the beta TGF biological activity wascarried out in the same way as with the CMDBS as described in Example 1ii. The incubation mixture contained 50 ng of beta TGF (in 0.1% bovineserum albumin) and trypsin (500 μg). Mesoglycan or sulodexide or dextransulfate or dextran or sucrase are used at the dose of 5000 μg.

The beta TGF biological activity is measured as described above after adilution of 50 times and by using CCL64 cells.

The results are presented in Table 4.

These results show that, like certain CMDBSs capable of responding tothe two selection criteria with regard to the FGFs and beta TGFs,sulodexide presents a significant protective activity for the beta TGFs.

c) Isolation of the heparan sulfate fraction of sulodexide andmesoglycan

Sulodexide and mesoglycan correspond to mixtures of several substancesessentially made up of different glycosaminoglycans (GAG).

By means of a first purification stage, it was established that a gramof dry product of each of these two products contained respectively 874mg for the mesoglycan and 795 mg for the sulodexide of total GAGs.

This purification was obtained by subjecting these solubilized productsto ion exchange chromatography (DEAE-Trisacryl) in order to remove allproteic contaminants. The total GAGs were then purified by eluting theDEAE gel with a sodium acetate solution, pH 4, containing 1.5M NaCl.

After an extensive dialysis phase against water, 60 mg of each productof GAG are digested by the ABC chondroitinase overnight at 37° C. (1unit per mg of GAG). This enzyme degrades all the GAGs with theexception of the heparan sulfates (HS). The products of digestion weresubjected to molecular sieve chromatography (G50 Sephadex, 1.8×95 cmcolumn). Elution was then carried out on ammonium bicarbonate buffer ata rate of 18 ml/hour. The non digested material corresponding to HS-typeGAGs was collected in the elution dead volume of the column.

The GAG concentrations were calculated from their uronic acid contentusing the carbazol method (Bitter T. and Muir H. M., 1962, Anal. Biochem4, pp. 330-334).

From these measurements the following composition of each of theproducts was obtained:

    ______________________________________                                                        Sulodexide                                                                            Mesoglycan                                            ______________________________________                                        Total GAGs        79%       87%                                               Heparan Sulfate Fraction (HS)                                                                   48%       52%                                               Other GAGs        31%       35%                                               ______________________________________                                    

The HS fractions of each of these two products were againchromatographed on a DEAE Trisacryl gel. 1 mg of each HS fraction,purified with mesoglycan (FIG. 6A) or sulodexide (FIG. 6B) in 3 ml, wasdeposited on a balanced column with 0.05M NaCl buffer, 0.05M TMS-Hcl pH7.5. After washing of the column with 10 volumes of the same bufferfollowed by washing with 10 volumes of 0.05M NaCl buffer, 0.05M ofsodium acetate pH 4, the material fixed to the column is desorbed by asaline gradient ranging from 0.05M NaCl to 1.5M NaCl in the same acetatebuffer. 1 ml of each fraction collected was measured by the carbazolemethod.

The material corresponding to the HS constituents of each of theoriginal products shows approximately the same elution profile and thusroughly the same apparent load. This elution peak maximum is obtainedfor a saline concentration of 0.94M NaCl. A defined fraction ofchondroitine sulfate (CSA) was subjected to the same protocol in orderto calibrate chromatography. This CSA fraction, containing only onesulfate group by disaccharide, is eluted at the ionic strength of 0.72MNaCl.

These results show that the HS fraction contains more sulfate groupsthan the reference CSAs. The HS fraction presents about two sulfategroups per dissacharidic unit.

These fractions were tested in order to discover their protective powerwith regard to the beta TGF and the FGF compared to the powersestablished with the respective raw products.

Semi-quantitative evaluation of the FGF protective effects of the FGF bydifferent polymers

As described above, a constant quantity of radioactive FGF is incubatedunder various conditions. After autoradiography of the reactionproducts, the non degraded quantity of radioactive FGF is quantified bydensitometry. The values correspond to the percentage of radio-labelledFGF found compared to the quantity deposited at the start of reaction(Table 5).

The results of Tables 4 and 5 show that the HSM and HSS fractions,originating respectively from mesoglycan and sulodexide, show protectiveeffects which are greater than these two compositions and are close to100%.

EXAMPLE 3

In vitro Inhibiting effects of CMDBSs and glycosaminoglycans on theactivity of leukocytic elastase and plasmin

The inhibiting powers of different CMDBS and of the intermediatecompounds of their synthesis were established for leukocytic elastaseand plasmin.

The purified leukocytic elastase was obtained by Elastin Products Co(Owenville, Mo., USA) and the plasmin from SIGMA.

The inhibition of enzymatic activities by these different compounds iscarried out at 37° C. in a thermostatic bath. The enzymes underconsideration are placed in solution in a 100 mM Tris-HCL buffer, pH 8for the elastase and pH 7.4 for the plasmin, in the presence of 0.02%sodium azide and 0.01% Triton X100 for the plasmin. The substrate andenzyme concentrations are: 0.10 mM MeO-Suc-Ala-Ala-Pro-Val-pNA(paranitroanilide) for the elastase at 8.3 nM, and 0.20 mMdVal-Leu-dLys-pNA for the plasmin at 77 nM. The IC₅₀ is determined foreach of the conditions.

Table 6 gives the results obtained, in which batch AM6 corresponds to aT40 dextran of 40,000 kD. Batch EM5 corresponds to a T10 dextran of10,000 kD. The intermediate products of synthesis are identified by thesymbols given above with an index number specifying the number of eachsubstitution reaction.

The IC₅₀ values show that the CMDBSs have non competitivehyperbolic-type inhibiting effects on leukocytic elastase activity whichare comparable to those of heparin, one of the best inhibitors of thisactivity (Ki of the order of 1 nM). In addition, and unlike heparin, theCMDBSs show inhibiting effects on plasmin.

Table 6 also shows that the inhibiting effects of the HSM and HSSfractions are greater than those of mesoglycan and sulodexide,respectively.

EXAMPLE 4

Healing effect of CMDBS on anastomosis of the colon

The batch of CMDBS used in this example is that described in Example 1.A 50 μg per ml solution of CMDBS is made in a physiological serum. MaleWistar rats (Wi/Wi, Ico, Iffa Credo France), weighing 250 to 300 g areanesthetized with sodic pentobarbital. A subumbilical midline laparotomyis performed over 1.5 cm. The rectocolic junction is individualized andthen sectioned with bistoury. The two banks of the anastomosis are thenimbibed with a solution containing either the CMDBS or a physiologicalbuffer for a period of two minutes. The anastomosis is carried outaccording to a termino-terminal mode in a plane by five separatestitches of polyglactine 910 thread, diameter 6/0. The knots are made of6 loops. Parietal closure is performed in two planes. The rats aredivided into two groups of 7 rats. The experiments are conducted indouble blind. Samples are taken on the 2nd, 4th and 7th days after theoperation. The quality of the healing is studied by measuring therupture pressure of the anastomosis, in millimeters of mercury. Thecolon is sectioned 2 cm on either side of the anastomosis, one end beingconnected to a self-pushing syringe which injects water at a low,constant rate, and the other end being linked to a manometer. Pressureis recorded continuously on a reel of paper. The rupture pressure isdetermined by analyzing the tracing. The values obtained are presentedin Table 7 below.

Results: At Day 2 (48 hours after the operation) a very markeddifference greater, by the order of three times, is measured between theanastomosis rupture forces realized with and without CMDBS treatment.This mean is statistically significant (p≦0.05). At Day 4 thisdifference is much smaller and corresponds to an increased resistance ofabout 10% in favor of the animals treated with CMDBS. At Day 7, there isno longer any difference between the treated and untreated resistances.The benefit of the CMDBS treatment is clearly apparent since it providesa greatly improved anastomosis solidity at the crucial moment, that isto say at the very start of healing, for the risks of anastomosisrupture always occur in the first few days.

EXAMPLE 5

Healing effect of the CMDBS on colitis induced by acetic acid

Groups of 6 Sprague Dawley male rats weighing approximately 200 g areplaced in hydric fasting 24 hours before the test. On the day of thetest, the animals are anesthetized with pentobarbital (30 mg/kg) byintraperitoneal route. After laparotomy, a colic instillation of 2 ml of5% acetic acid is performed 2 cm from the caecum. The acetic acid isleft in contact for 10 seconds and the colon is then rinsed by 3 ml ofsaline phosphate buffer (PBS) at pH 7. The wound is then closed. Theanimals are sacrificed 72 hours later by cervical elongation. The colicalterations are noted on the following scale:

0: normal colon

1: congestion

2: superficial necrosis of the mucosa

4: major necrosis of the mucosa with edema

5: perforation

Treatment

A first control group of six rats does not receive treatment.

A second group receives 50 μg of an aqueous solution of CMDBS (batchAM26), administered orally morning and evening for three days.

A third group receives sucralfate (marketed under the name of ULCARPharmaceutical Laboratories Fabre). The supplier's recommended dose isused (500 mg/kg, i.e. 100 mg per rat) administered orally twice a dayfor three days.

Statistical analysis

The mean and the standard error were calculated for each batch ofanimals. Statistical comparison is performed using White's nonparametric test compared to the control.

The results are presented in Table 8 below.

As these results show, the CMDBS reduces the seriousness of the coliclesions induced by acetic acid by about 75% (significant effect,p≦0.05). In contrast, the sucralfate has no significant effect on thehealing of these ulcers.

EXAMPLE 7

HBGPP-based compositions

In examples 5 and 6 described above, the CMDBS is a buffered salineaqueous solution (PBS buffer) or physiological serum at concentrationsof 50 μg/ml. Administration is local or per os; it may be intravenous.Other compositions may be proposed provided that the dose of CMDBSreaching the site of the lesion lies between 1 μg and 1000 μg.

                  TABLE 1                                                         ______________________________________                                        Protective effects of CMDBS and heparin                                       against beta TGF degradation by trypsin                                       incubation mixture at 37° C. for 10                                    min and containing, per milliliter,                                                             % inhibiting activity of the                                according to the indication: CMDBS                                                              incorporation of tritiated thymidine                        or heparin (5000 μg): βTGF (50                                                          in CCL64 cells (after dilution 50                           ng): trypsin (500 μg)                                                                        times of incubation mixture)                                ______________________________________                                        Incubation buffer only                                                                          0                                                           CMDBS (5000 μg)                                                                              0                                                           Heparin (5000 μg)                                                                            0                                                           Trypsin (1000 μg)                                                                            0                                                           Beta TGF (50 ng)  100                                                         Beta TGF + CMDBS (batch AM26)                                                                   100                                                         Beta TGF + heparin                                                                              100                                                         Beta TGF + trypsin                                                                              5                                                           Beta TGF + CMDBS + trypsin                                                                      75                                                          Beta TGF + heparin + trypsin                                                                    10                                                          ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Non inhibiting effect of CMDBS on trypsin                                     ______________________________________                                        Trypsin (10 ug/ml) + S87                                                                            100                                                     Trypsin + S87 + 5 μg/ml CMDBS                                                                    100                                                     Trypsin + S87 + 50 μg/ml CMDBS                                                                   100                                                     Trypsin + S87 + 500 μg/ml CMDBS                                                                  100                                                     Trypsin + S87 + STBI   0                                                      ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Origin, anticoagulant activity and partial composition of mesoglycan          and sulodexide (supplier's information)                                                     Sulodexide                                                                             Mesoglycan                                             ______________________________________                                        Origin          pig duodenum                                                                             Aorta                                              Anticoagulant activity                                                                        50-70 IU/mg                                                                              <50 IU/mg                                          Chemical composition                                                          Dermatan sulfate                                                                              20-35%     25-60%                                             Chondroitine sulfate                                                                          2-7%        3-15%                                             Heparan sulfate +          +                                                  ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Protection of the beta TGF by various polymers                                ______________________________________                                        Beta TGF              100%                                                    Beta TGF + trypsin     0%                                                     Beta TGF + mesoglycan 100%                                                    Beta TGF + mesoglycan + trypsin                                                                      50%                                                    Beta TGF + HSM        100%                                                    Beta TGF + HSM + trypsin                                                                             75%                                                    Beta TGF + sulodexide 100%                                                    Beta TGF + sulodexide + trypsin                                                                      20%                                                    Beta TGF + HSS        100%                                                    Beta TGF + HSS + trypsin                                                                             45%                                                    Beta TGF + dextran    100%                                                    Beta TGF + dextran + trypsin                                                                         0%                                                     Beta TGF + dextran sulfate                                                                          100%                                                    Beta TGF + dextran sulfate + trypsin                                                                 0%                                                     Beta TGF + sucrase    100%                                                    Beta TGF + sucrase + trypsin                                                                         0%                                                     ______________________________________                                         HSM = Heparan sulfates purified from mesoglycan                               HSS = Heparan sulfates purified from sulodexide                          

                  TABLE 5                                                         ______________________________________                                        Protection of the FGF by various polymers                                     ______________________________________                                                            PROTECTION (in %)                                         ______________________________________                                        FGF alone             100%                                                    FGF + trypsin         0%                                                      FGF + trypsin + heparin                                                                             100%                                                    FGF + trypsin + mesoglycan                                                                          75%                                                     FGF + trypsin + sulodexide                                                                          70%                                                     FGF + trypsin + heparinase treated mesoglycan                                                       0%                                                      FGF + trypsin + heparinase treated sulodexide                                                       0%                                                      FGF + trypsin + heparinase treated heparin                                                          0%                                                      FGF + HSM + trypsin   95%                                                     FGF + HSS + trypsin   90%                                                     ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        Inhibition of elastase and plasmin activities                                                Leukocytic elastase                                                                        plasmin                                           Tested compounds                                                                             IC.sub.50 in μg/ml                                                                      IC.sub.50 in μg/ml                             ______________________________________                                        CMDBS, batch AM6                                                                             2.2          1.5                                               T40            >100         >100                                              CMDBS, batch EM5                                                                             10           7                                                 T10 CMD2B      50           53                                                T10 5CMD1B     >100         >100                                              T10 3CMD       >100         >100                                              T10            >100         >100                                              Mesoglycan     72           65                                                HS mesoglycan  20           22                                                Sulodexide     79           75                                                HS sulodexide  25           20                                                Heparin        1.8                                                            Lipo-heparin                0.5                                               ______________________________________                                         HSM = Heparan sulfates purified from mesoglycan                               HSS = Heparan sulfates purified from sulodexide                          

                  TABLE 7                                                         ______________________________________                                        Study of the healing of the rectocolic junction                                        DAY 2        DAY 4                                                            CONTROLS                                                                              CMDBS    CONTROLS  CMDBS                                     ______________________________________                                        Rupture pressure in                                                                      38        48       112     136                                     mm of Hg   20        96       140     140                                                18        76       140     148                                                35        53       121     144                                                20        82       98      156                                                18        66       108                                                                  50                                                       mean       22.45     67.28    119.8   144.8                                   S.D.       8.33      16.87    15.77   6.88                                    n          6         7        6       5                                       ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        Effect of CMDBS on colitis induced by acetic acid                             Treatment         Alteration score                                            ______________________________________                                        Controls          2.83 ± 0.48                                              CMDBS 50 μg/rat per os                                                                       0.70 ± 0.37                                              Sucralfate 100 mg/rat per os                                                                    2.25 ± 0.48                                              ______________________________________                                    

We claim:
 1. A method of treating a lesion of the digestive tract or ofprimary or secondary derived tissue of the endoderm or the mesoderm,which comprises administering to a human or other animal in need of suchtreatment an effective amount of a pharmaceutical composition containinga) at least one polymer or one biopolymer, called HBGFPP, with theexception of mesoglycan, specifically protecting the growth factors offamilies of FGFs and beta TGFs from tryptic degradation and notsignificantly inhibiting coagulation, in association with b) at leastone pharmacologically acceptable excipient.
 2. A method according toclaim 1, wherein the polymer or biopolymer presents an anticoagulantactivity of less than 50 international units per mg of polymer.
 3. Amethod according to claim 1, wherein the said polymer does notsubstantially activate the complement system.
 4. A method according toclaim 1, wherein the said polymer potentializes in vitro the FGF.
 5. Amethod according to one of claim 1, wherein the said polymersubstantially inhibits the proteasic activities of elastase and/orplasmin.
 6. A method according to one of claim 1, wherein the saidpolymer or biopolymer is a polysaccharide.
 7. A method according toclaim 6, wherein the said polysaccharide is primarily composed ofglucose residues.
 8. A method according to claim 6, wherein thepolysaccharide comprises glucosamine and/or uronic acid residues.
 9. Amethod according to claim 8, wherein the polysaccharide comprisesglucosamine-uronic acid dimers.
 10. A method according to claim 8,wherein the said polysaccharide is a glycosaminoglycan, or a sulfate ofone of these compounds, possibly associated with a lipid, a peptide or aprotide.
 11. A method according to claim 1, wherein the saidpolysaccharide is a substituted dextran.
 12. A method according to claim11, wherein the said polysaccharide is a CMDBS.
 13. A method accordingto claim 1, wherein the said polymer is of non-osidic nature.
 14. Amethod according to claim 1 wherein a) is the sole essential activecomponent for treating a lesion of the digestive tract or lesion ofprimary or secondary derived tissue of the endotherm or of the mesoderm.